Brain cells communicate with each other using synapses. Typically, several thousand chemical synapses converge onto a single postsynaptic neuron. The computational power of circuits in the central nervous system (CNS) relies critically on an abundance of synapses and has thus benefitted from their extreme miniaturization. However, the ~1 micrometer size of typical CNS synapses poses great challenges for their cell biology and for investigators interested in their inner workings. Tiny nerve terminals function with only a limited number of vesicles, as few as ~30 in the most studied model, hippocampal synapses in culture. Following vesicle fusion and exocytosis of neurotransmitter, recycling of vesicular membrane is critical to allow transmission to continue during extended periods of intermittent bursting activity typical of hippocampal neurons. Classical full-collapse fusion (FCF) proceeds by vesicles completely flattening into the plasma membrane, thereby releasing their transmitter, but also completely losing their lipid and protein content and spherical shape. Vesicles are then reconstructed via clathrin-mediated membrane retrieval. Another set of phenomena generically known as kiss and run (K&R) has been proposed wherein vesicular identity is maintained and the same vesicle is repeatedly reused. The likely consequence of K&R is increased synaptic efficiency and information throughput. To gain further insights into how vesicles meet the demands of intense activity, and balance use of distinct fusion modes, the following specific aims are proposed. In Aim 1, we will employ novel optical approaches to study the relative weights of K&R and FCF. Vesicular uptake of a newly created pH-sensing nanoparticle, comprising multiple pH sensor molecules attached to a single quantum dot (Qdot), will allow us to assess the prevalence of K&R and reuse in whole presynaptic terminals. The pH signals from single vesicles, and possibly signals reflecting chloride and glutamate concentrations, will provide insights into how vesicles reacidify and refill themselves with excitatory neurotransmitter. In Aim 2, we will use a newly constructed 3-D microscope to track the movements of individual vesicles marked with a single Qdot. We will determine whether the pre-fusion movements of vesicles provide tell-tale indications of which mode of fusion they will employ. We will ask if post-fusion movements support the notion that vesicles undergoing K&R are reused again at or near the same location (kiss-and-stay). Finally, in Aim 3, we will track the motion of the vesicles that have the highest chance of fusing, the so-called readily releasable pool, to see whether they reside closer to the fusion site than other vesicles. Do such vesicles bear a memory of their pool of origin that survives FCF and clathrinmediated retrieval, so they somehow go to the head of line in a second round of neurotransmission? If so, we will press on to evaluate specific candidates for the molecular tag that confers this persistent pool identity. These experiments will advance methods that bridge the gap between light and electron microscopy and improve our currently imperfect understanding of the fundamental process of neurotransmission.